1. Technical Field
This application relates to proteins which are involved in the growth, regulation or maintenance of nervous tissue, particularly neurons. In particular, it relates to NGF variants that have activities of another neurotrophic factor NT-3. NGF variants which have trkC-binding activity and trkC-signal inducing activity are provided. The variants optionally have trkA or trkB binding and signal induction activity. The NGF variants of the present invention are useful in the treatment of neuronal disorders. Nucleic acids and expression vectors encoding the NGF variant neurotrophins are also provided.
2. Introduction
The survival and maintenance of differentiated function of vertebrate neurons is influenced by the availability of specific proteins referred to as neurotrophins. The neurotrophins form a highly homologous family of growth factors that are important for survival and maintenance of neurons during developmental and adult stages of the vertebrate nervous system (for review see 99). Limited production of neurotrophins results in death of superfluous neurons (for reviews, see (1); (2)). The various neurotrophins differ functionally in their ability to support survival of distinct neuronal populations in the central and the peripheral nerve system (3), (4); (5), (80).
The neurotrophin family is a highly homologous family which includes NT-3 (6, 7, 5, 8, 9, 10), nerve growth factor (NGF) (11, 12), brain-derived neurotrophic factor (BDNF) (13, 14) and neurotrophin 4/5 (NT-4/5) (15, 16, 17) and neurotrophin-6 (NT-6) (91, 92).
Studies suggest that neurotrophins transduce intracellular signaling at least in part through the ligand-dependent activation of a class of tyrosine kinase-containing receptors of Mr=140-145,000 known as the trks (18); (19) (21); (20) (22); (23); (24); (25); (26). Binding of the neurotrophins induces autophosphorylation of the trk receptors which triggers the subsequent steps in the signal transduction cascade (95). Thus, the signal transduction pathway of neurotrophins is initiated by this high-affinity binding to and activation of specific tyrosine kinase receptors and subsequent receptor autophosphorylation (19); (27). Although there is some degree of cross-receptor interaction between the neurotrophins and the different trks, the predominant specificity appears to be NGF/trkA, BDNF/trkB, and NT-3/trkC while NT-4/5 appears to interact primarily with trkB as efficiently as BDNF (27); (19) (21); (25); (22); (28); (18); (28a). NGF interacts exclusively with trkA (21) while BDNF and NT-4/5 bind to trkB (29). TrkA and trkB can respond in vitro under certain circumstances to multiple neurotrophins (6); (23). TrkC responds exclusively to NT-3 (25); (26). NT-3 signals preferably through trkC but can also bind to trkA and trkB with lower affinity (25; 101) (FIG. 1). Thus, the most stringent member of the trk receptors in terms of specificity (trkC) interacts exclusively with the most promiscuous ligand (NT-3) of the neurotrophin family.
However, the neuronal environment does restrict trkA and trkB in their ability to respond to non-preferred neurotrophic ligands (29). In addition to the trk family of receptors, the neurotrophins can also bind to a different class of receptor termed the p75 low affinity NGF receptor (p75; (30); (31)) which has an unknown mechanism of transmembrane signaling but is structurally related to a receptor gene family which includes the tumor necrosis factor receptor (TNFR), CD40, 0X40, and CD27 (32); (33); (34), (35); (36); (37)). The role of the gp75 in the formation of high-affinity binding sites and in the signal transduction pathway of neurotrophins is as yet unclear (for reviews see (38); (39)).
An examination of the primary amino acid sequence of the neurotrophins reveals seven regions of 7-10 residues each which account for 85% of the sequence divergence among the family members.
Nerve growth factor (NGF) is a 120 amino acid polypeptide homodimeric protein that has prominent effects on developing sensory and sympathetic neurons of the peripheral nervous system. NGF acts via specific cell surface receptors on responsive neurons to support neuronal survival, promote neurite outgrowth, and enhance neurochemical differentiation. NGF actions are accompanied by alterations in neuronal membranes (40), (41), in the state of phosphorylation of neuronal proteins (42), (43), and in the abundance of certain mRNAs and proteins likely to play a role in neuronal differentiation and function (see, for example (44)).
Forebrain cholinergic neurons also respond to NGF and may require NGF for trophic support. (45). Indeed, the distribution and ontogenesis of NGF and its receptor in the central nervous system (CNS) suggest that NGF acts as target-derived neurotrophic factor for basal forebrain cholinergic neurons (46), (81).
NT-3 transcription has been detected in a wide array of peripheral tissues (e.g. kidney, liver, skin) as well as in the central nerve system (e.g. cerebellum, hippocampus) (5); (7), (82). During development, NT-3 mRNA transcription is most prominent in regions of the central nervous system in which proliferation, migration and differentiation of neurons are ongoing (50). Supporting evidence for a role in neuronal development includes the promoting effect of NT-3 on neural crest cells (51) and the stimulation of the proliferation of oligodendrocyte precursor cells in vivo (79). NT-3 also supports in vitro the survival of sensory neurons from the nodose ganglion (NG) (7); (5), (83) and a population of muscle sensory neurons from dorsal root ganglion (DRG) (52). In addition to these in vitro studies, a recent report showed that NT-3 prevents in vivo the degeneration of adult central noradrenergic neurons of the locus coerulus in a model that resembles the pattern of cell loss found in Alzheimer's disease.
Extensive mutational analyses of human NT-3 (101) and mouse and human NGF (55; 98) suggested the binding sites for trkC and trkA, respectively. The three-dimensional structures of several neurotrophins have been resolved by X-ray crystallography (59; 93; 96). In NGF the N-terminal residues contribute significantly to affinity for trkA (98) and provide the most important determinants for specificity (55; 101). Significant losses of biological activity and receptor binding were observed with purified homodimers of human and mouse NGF, representing homogenous truncated forms modified at the amino and carboxy termini (47); (48); (49). The 109 amino acid species (10-118)hNGF, resulting from the loss of the first 9 residues of the N-terminus and the last two residues from the C-terminus of purified recombinant human NGF, is 300-fold less efficient in displacing mouse [125I]NGF from the human trkA receptor compared to (1-118)hNGF (49). It is 50- to 100-fold less active in dorsal root ganglion and sympathetic ganglion survival compared to (1-118)hNGF (48). The (10-118)hNGF has been reported to have considerably lower trkA tyrosine kinase autophosphorylation activity (49).
For NT-3 it has been demonstrated that the epitope for trkC is formed by residues in the central β-strand bundle region but does not include residues from non-conserved loops or the first six residues of the N-terminus (101). However, a non-conserved β-hairpin loop encompassing residues 40-49 (NGF residue numbers will be used throughout the text) has been proposed to mediate trkA/trkC specificity (94), though this loop does not contribute to NT-3 binding to trkC (101). The mechanism of trkC discrimination, however, is unclear, especially since the most important residue in NT-3 involved in binding to trkC, R103, is conserved in all neurotrophins.
The elucidation of the structural determinants for neurotrophin specificity is important for understanding the function and evolution of this family of growth factors. Furthermore, administration of neurotrophins in models of nerve lesions have been shown to be beneficial for regeneration and survival of neurons (97; 103). Since the neurotrophins have become candidates for therapeutics for a variety of neurodegenerative diseases, knowledge of the structural mechanism of neurotrophic specificity and function will help develop novel neurotrophin-based therapeutics.
There have been some limited attempts to create chimeric or pan-neurotrophic factors. (See (53); (56); (54), (55)). Neuronal populations involved in neurodegenerative disorders may express more than one trk receptor and therefore administration of molecules with multiple specificities, such as MNTS-1 (101) or PNT-1 (55) could be advantageous compared to administration of a single monospecific neurotrophin or a cocktail of monospecific neurotrophins. For example, the various members of the neurotrophin family may have different pharmacokinetics and therefore the behavior of neurotrophin cocktails could be difficult to predict or control.
There is a need for neurotrophic molecules that have more than one neurotrophin activity and/or have improved pharmacokinetic properties and that are readily administered and retain effectiveness. These and other advantages are provide by the molecules and methods presented herein.